Compositions and methods for modulation of ikbkap splicing

ABSTRACT

The present disclosure provides compounds comprising oligonucleotides complementary to a portion of the IKBKAP gene. Certain such compounds are useful for hybridizing to a portion of the IKBKAP gene, including but not limited to a portion of the IKBKAP gene in a cell. In certain embodiments, such hybridization results in modulation of splicing of the IKBKAP gene. In certain embodiments, the IKBKAP gene includes a mutation that results in defective splicing and a truncated IKAP protein. In certain embodiments, hybridization of oligonucleotides complementary to a portion of the IKBKAP gene results in a decrease in the amount of defective splicing and truncated IKAP protein. In certain embodiments, hybridization of oligonucleotides complementary to a portion of the IKBKAP gene results in an increase in the amount of normal splicing and functional, full-length IKAP protein. In certain embodiments, oligonucleotides are used to treat Familial Dysautonomia.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0100WOSEQ.txt, created Jan. 9, 2013, which is 96 kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND

Newly synthesized eukaryotic mRNA molecules, also known as primarytranscripts or pre-mRNA, made in the nucleus, are processed before orduring transport to the cytoplasm for translation. Processing of thepre-mRNAs includes addition of a 5′ methylated cap and an approximately200-250 base poly(A) tail to the 3′ end of the transcript.

Another step in mRNA processing is splicing of the pre-mRNA, whichoccurs in the maturation of 90-95% of mammalian mRNAs. Introns (orintervening sequences) are regions of a primary transcript (or the DNAencoding it) that are not included in the coding sequence of the maturemRNA. Exons are regions of a primary transcript that remain in themature mRNA when it reaches the cytoplasm. The exons are splicedtogether to form the mature mRNA sequence. Splice junctions are alsoreferred to as splice sites with the junction at the 5′ side of theintron often called the “5′ splice site,” or “splice donor site” and thejunction at the 3′ side of the intron called the “3′ splice site” or“splice acceptor site.” In splicing, the 3′ end of an upstream exon isjoined to the 5′ end of the downstream exon. Thus the unspliced RNA (orpre-mRNA) has an exon/intron junction at the 5′ end of an intron and anintron/exon junction at the 3′ end of an intron. After the intron isremoved, the exons are contiguous at what is sometimes referred to asthe exon/exon junction or boundary in the mature mRNA. Cryptic splicesites are those that are not used in wild-type pre-mRNA, but may be usedwhen the natural splice site is inactivated or weakened by mutation, orin conjunction with a mutation that creates a new splice site elsewhereon the pre-mRNA. Alternative splicing, defined as the splicing togetherof different combinations of exons or exon segments, often results inmultiple mature mRNA transcripts expressed from a single gene.

Up to 50% of human genetic diseases resulting from a point mutation arecaused by aberrant splicing. Such point mutations can either disrupt acurrent splice site or create a new splice site, resulting in mRNAtranscripts comprised of a different combination of exons or withdeletions in exons. Point mutations also can result in activation of acryptic splice site(s), disrupt a branch site (which functions during anintermediate step in splicing catalysis) or disrupt regulatory ciselements (i.e., splicing enhancers or silencers, which can be created,destroyed, strengthened or weakened by mutation) (Cartegni et al., Nat.Rev. Genet., 2002, 3, 285-298; Crawczak et al., Hum. Genet., 1992, 90,41-54).

Antisense oligonucleotides have been used to target mutations that leadto aberrant splicing in several genetic diseases in order to redirectsplicing to give a desired splice product (Kole, Acta BiochimicaPolonica, 1997, 44, 231-238). Such diseases include β-thalassemia(Dominski and Kole, Proc. Natl. Acad. Sci. USA, 1993, 90, 8673-8677;Sierakowska et al., Nucleosides & Nucleotides, 1997, 16, 1173-1182;Sierakowska et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 12840-44;Lacerra et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 9591-9596);dystrophy Kobe (Takeshima et al., J. Clin. Invest., 1995, 95, 515-520);Duchenne muscular dystrophy (Dunckley et al. Nucleosides & Nucleotides,1997, 16, 1665-1668; Dunckley et al. Human Mol. Genetics, 1998, 5,1083-90); osteogenesis imperfecta (Wang and Marini, J. Clin Invest.,1996, 97, 448-454); and cystic fibrosis (Friedman et al., J. Biol.Chem., 1999, 274, 36193-36199).

Antisense compounds have also been used to alter the ratio of the longand short forms of Bcl-x pre-mRNA (U.S. Pat. No. 6,172,216; U.S. Pat.No. 6,214,986; Taylor et al., Nat. Biotechnol. 1999, 17, 1097-1100) orto force skipping of specific exons containing premature terminationcodons (Wilton et al., Neuromuscul. Disord., 1999, 9, 330-338). U.S.Pat. No. 5,627,274 and WO 94/26887 disclose compositions and methods forcombating aberrant splicing in a pre-mRNA molecule containing a mutationusing antisense oligonucleotides which do not activate RNAse H.

Antisense compounds targeting splicing-inhibitory elements in exons ortheir flanking introns have also been used to increase the use of suchexons during splicing, e.g., in the context of spinal muscular atrophy(Cartegni Nat Struct Biol; Imaizumi; Hua PLoS Biol; Singh; other Hua etal papers, etc.).

Familial dysautonomia (FD), a rare genetic disorder found almostexclusively in the Ashkenazi Jewish population, is an autosomalrecessive condition that is caused by a single intronic point mutationin intron 20 (IVS20+6T→C) of the IKBKAP gene (Maayan, C., Kaplan, E.,Shachar, S., Peleg, O., and Godfrey, S. 1987, “Incidence of familialdysautonomia in Israel 1977-1981,” Clin Genet. 32:106-108; Slaugenhaupt,S. A., and Gusella, J. F. 2002, “Familial dysautonomia,” Curr Opin GenetDev 12:307-311; Anderson, S. L., Coli, R., Daly, I. W., Kichula, E. A.,Rork, M. J., Volpi, S. A., Ekstein, J., and Rubin, B. Y. 2001, “Familialdysautonomia is caused by mutations of the IKAP gene,” Am J Hum Genet.68:753-758). FD, also known as Riley-Day syndrome and hereditary sensoryautonomic neuropathy type-III (HSAN-III), is characterized by poordevelopment and progressive degeneration of sensory and autonomicneurons. Notable symptoms include anhidrosis, decreased taste, depresseddeep tendon reflexes, postural hypotension, loss of pain and temperatureperception, alacrima, gastroesophageal reflux, and scoliosis (Axelrod,F. B., and Simson, G. G. V. 2007 “Hereditary sensory and autonomicneuropathies: types II, III, and IV,” Orphanet Journal of Rare Diseases2:). The extent and severity of the symptoms vary among patients, buteven with advanced management, the disease leads to premature death,with only half of the patients surviving to 40 years of age.

Antisense technology is an effective means for modulating the expressionof one or more specific gene products, including alternative spliceproducts, and is uniquely useful in a number of therapeutic, diagnostic,and research applications. The principle behind antisense technology isthat an antisense compound, which hybridizes to a target nucleic acid,modulates gene expression activities, such as transcription, splicing ortranslation, through one of a number of antisense mechanisms. Thesequence specificity of antisense compounds makes them extremelyattractive as tools for target validation and gene functionalization, aswell as therapeutics to selectively modulate the expression of genesinvolved in disease.

SUMMARY

In certain embodiments, the present disclosure provides compoundscomprising oligonucleotides. In certain embodiments, sucholigonucleotides are complementary to an IKBKAP transcript. In certainsuch embodiments, the oligonucleotide is complementary to a targetregion of the IKBKAP transcript comprising exon 20, intron 19, andintron 20. In certain embodiments, the IKBKAP transcript comprises amutation that results in an aberrant splice site. In certainembodiments, the IKBKAP transcript comprises a mutation that results inthe exclusion of exon 20 from the mature IKBKAP mRNA. In certainembodiments, oligonucleotides inhibit aberrant splicing of a mutantIKBKAP transcript. In certain such embodiments, normal splicing of theIKBKAP transcript is increased. In certain embodiments, functional IKAPprotein having exon 20 is increased. In certain embodiments, functionalIKAP protein having exons 20-37 is increased.

The present disclosure provides the following non-limiting numberedembodiments:

Embodiment 1

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising at least8 contiguous nucleobases complementary to a target region of equallength of an IKBKAP transcript.

Embodiment 2

The compound of embodiment 1, wherein nucleobase sequence comprises atleast 8 contiguous nucleobases complementary to intron 19, intron 20, orexon 20 of an IKBKAP transcript.

Embodiment 3

The compound of any of embodiments 1 to 2, wherein the modifiedoligonucleotide is 12 to 20 nucleosides in length.

Embodiment 4

The compound of any of embodiments 1 to 2, wherein the modifiedoligonucleotide is 14 to 16 nucleosides in length.

Embodiment 5

The compound of any of embodiments 1 to 2, wherein the oligonucleotideis 12 nucleosides in length.

Embodiment 6

The compound of any of embodiments 1 to 2, wherein the oligonucleotideis 14 nucleosides in length.

Embodiment 7

The compound of any of embodiments 1 to 2, wherein the oligonucleotideis 15 nucleosides in length.

Embodiment 8

The compound of any of embodiments 1 to 2, wherein the oligonucleotideis 16 nucleosides in length.

Embodiment 9

The compound of any of embodiments 1 to 2, wherein the oligonucleotideis 20 nucleosides in length.

Embodiment 10

The compound of any of embodiments 1 to 10 having a nucleobase sequencecomprising at least 9 contiguous nucleobases complementary to a targetregion of equal length of an IKBKAP transcript.

Embodiment 11

The compound of any of embodiments 1 to 10 having a nucleobase sequencecomprising at least 10 contiguous nucleobases complementary to a targetregion of equal length of an IKBKAP transcript.

Embodiment 12

The compound of any of embodiments 1 to 10 having a nucleobase sequencecomprising at least 11 contiguous nucleobases complementary to a targetregion of equal length of an IKBKAP transcript.

Embodiment 13

The compound of any of embodiments 1 to 10 having a nucleobase sequencecomprising at least 12 contiguous nucleobases complementary to a targetregion of equal length of an IKBKAP transcript.

Embodiment 14

The compound of any of embodiments 1 to 6 having a nucleobase sequencecomprising at least 13 contiguous nucleobases complementary to a targetregion of equal length of an IKBKAP transcript.

Embodiment 15

The compound of any of embodiments 1 to 6 having a nucleobase sequencecomprising at least 14 contiguous nucleobases complementary to a targetregion of equal length of an IKBKAP transcript.

Embodiment 16

The compound of any of embodiments 1 to 4 or 7 having a nucleobasesequence comprising at least 15 contiguous nucleobases complementary toa target region of equal length of an IKBKAP transcript.

Embodiment 17

The compound of any of embodiments 1 to 4 or 8 having a nucleobasesequence comprising at least 16 contiguous nucleobases complementary toa target region of equal length of an IKBKAP transcript.

Embodiment 18

The compound of any of embodiments 1 to 4 or 9 having a nucleobasesequence comprising at least 17 contiguous nucleobases complementary toa target region of equal length of an IKBKAP transcript.

Embodiment 19

The compound of any of embodiments 1 to 4 or 10 having a nucleobasesequence comprising at least 18 contiguous nucleobases complementary toa target region of equal length of an IKBKAP transcript.

Embodiment 20

The compound of any of embodiments 1 to 19, wherein the modifiedoligonucleotide comprises at least one modified nucleoside.

Embodiment 21

The compound of any of embodiments 1 to 20, wherein at least onemodified nucleoside comprises a modified sugar moiety.

Embodiment 22

The compound of embodiment 21, wherein at least one modified sugarmoiety is a 2′-substituted sugar moiety.

Embodiment 23

The compound of embodiment 22, wherein the 2′-substitutent of at leastone 2′-substituted sugar moiety is selected from the group consisting of2′-OMe, 2′-F, and 2′-MOE.

Embodiment 24

The compound of embodiment 23, wherein the 2′-substituent of at leastone 2′-substituted sugar moiety is a 2′-MOE.

Embodiment 25

The compound of any of embodiments 1 to 21, wherein at least onemodified sugar moiety is a bicyclic sugar moiety.

Embodiment 26

The compound of embodiment 25, wherein at least one bicyclic sugarmoiety is LNA or cEt.

Embodiment 27

The compound of any of embodiments 1 to 21, wherein at least one sugarmoiety is a sugar surrogate.

Embodiment 28

The compound of embodiment 27, wherein at least one sugar surrogate is amorpholino.

Embodiment 29

The compound of embodiment 27, wherein at least one sugar surrogate is amodified morpholino.

Embodiment 30

The compound of any of embodiments 1 to 29, wherein each nucleoside ofthe modified oligonucleotide is a modified nucleoside, eachindependently comprising a modified sugar moiety.

Embodiment 31

The compound of any of embodiments 1 to 29, wherein the modifiedoligonucleotide comprises at least two modified nucleosides comprisingmodified sugar moieties that are the same as one another.

Embodiment 32

The compound of any of embodiments 1 to 29, wherein the modifiedoligonucleotide comprises at least two modified nucleosides comprisingmodified sugar moieties that are different from one another.

Embodiment 33

The compound of any of embodiments 1 to 30, wherein each nucleoside ofthe modified oligonucleotide is a modified nucleoside.

Embodiment 34

The compound of any of embodiments 1 to 30, wherein each nucleoside ofthe modified oligonucleotide is a modified nucleoside, and each modifiednucleoside comprises the same modification.

Embodiment 35

The compound of embodiment 34, wherein the modified nucleosides eachcomprise the same 2′-substituted sugar moiety.

Embodiment 36

The compound of embodiment 35, wherein the 2′-substituted sugar moietyis selected from 2′-F, 2′-OMe, and 2′-MOE.

Embodiment 37

The compound of embodiment 36, wherein the 2′-substituted sugar moietyis 2% MOE.

Embodiment 38

The compound of any of embodiments 1 to 37, wherein at least oneinternucleoside linkage is a modified internucleoside linkage.

Embodiment 39

The oligonucleotide of any of embodiments 1 to 38, wherein eachinternucleoside linkage is a modified internucleoside linkage.

Embodiment 40

The compound of any of embodiments 1 to 39, wherein the modifiedinternucleoside linkage is phosphorothioate.

Embodiment 41

The compound of any of embodiments 1 to 40, wherein the oligonucleotideis targeted to an intronic splicing silencer element.

Embodiment 42

The compound of any of embodiments 1 to 40, wherein the oligonucleotideis targeted to an exonic splicing silencer element.

Embodiment 43

The compound any of embodiments 1 to 40, wherein nucleobase sequencecomprises at least 8 contiguous nucleobases complementary to intron 19,intron 20, or exon 20 of a nucleic acid molecule encoding IKAP.

Embodiment 44

The compound of any of embodiments 1 to 40, wherein the oligonucleotideis targeted to intron 19.

Embodiment 45

The compound of any of embodiments 1 to 40, wherein the oligonucleotideis targeted to intron 20.

Embodiment 46

The compound of any of embodiments 1 to 40, wherein the oligonucleotideis targeted to exon 20.

Embodiment 47

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 34622 to 34895 of SEQ ID NO: 1.

Embodiment 48

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 34622 to 34721 of SEQ ID NO: 1.

Embodiment 49

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 34722 to 34795 of SEQ ID NO: 1.

Embodiment 50

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 34796 to 34881 of SEQ ID NO: 1.

Embodiment 51

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 34722 to 34795 of SEQ ID NO: 1.

Embodiment 52

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 34801 to 34828 of SEQ ID NO: 1.

Embodiment 53

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 34801 to 34826 of SEQ ID NO: 1.

Embodiment 54

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 34802 to 34821 of SEQ ID NO: 1.

Embodiment 55

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, or 68.

Embodiment 56

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 60.

Embodiment 57

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 61.

Embodiment 58

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 62.

Embodiment 59

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 63.

Embodiment 60

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 64.

Embodiment 61

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 65.

Embodiment 62

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprises an at least 8 nucleobaseportion of SEQ ID NO: 66.

Embodiment 63

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 67.

Embodiment 64

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 68.

Embodiment 65

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 40, 41, 42, 43, or 44.

Embodiment 66

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 40.

Embodiment 67

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 41.

Embodiment 68

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 42.

Embodiment 69

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 43.

Embodiment 70

The compound of any of embodiments 1 to 46, wherein the oligonucleotidecomprises a nucleobase sequence comprising an at least 8 nucleobaseportion of SEQ ID NO: 44.

Embodiment 71

A pharmaceutical composition comprising the compound of any ofembodiments 1 to 70, and a pharmaceutically acceptable carrier ordiluent.

Embodiment 72

A method of modulating splicing in an IKBKAP transcript in a cellcomprising contacting the cell with a compound according to any ofembodiments 1 to 70.

Embodiment 73

A method of increasing inclusion of exon 20 in an IKBKAP transcript,comprising contacting a cell with the compound of any of embodiments 1to 70.

Embodiment 74

A method of increasing functional IKAP protein in a cell, comprisingcontacting the cell with a compound according to any of embodiments 1 to70.

Embodiment 75

A method of increasing IKAP protein having amino acids encoded by exons20-37 in a cell, comprising contacting the cell with a compoundaccording to any of embodiments 1 to 70.

Embodiment 76

A method for treating a condition characterized at least in part bydefective splicing of an IKBKAP transcript, comprising administering atherapeutically effective amount of the compound of any of embodiments 1to 70, to a subject in need thereof.

Embodiment 77

Use of the compound of any of embodiments 1 to 70 for the preparation ofa medicament for increasing inclusion of exon 20 in an IKBKAPtranscript.

Embodiment 78

Use of the compound of any of embodiments 1 to 70 for the preparation ofa medicament for the treatment of Familial Dysautonomia.

Embodiment 79

A compound of any of embodiments 1 to 70 for use in treating FamilialDysautonomia.

Embodiment 80

The method of any of embodiments 72 to 76, wherein the antisensecompound is administered into the central nervous system.

Embodiment 81

The method of any of embodiments 72 to 76, wherein the antisensecompound is administered systemically.

Embodiment 82

Then method of any of embodiments 72 to 76, wherein the systemicadministration is by intravenous or intraperitoneal injection.

Embodiment 83

Then method of any of embodiments 72 to 76, wherein the systemicadministration is by introcerebroventricular injection.

Embodiment 84

The method of any of embodiments 72 to 76, wherein the systemicadministration and the administration into the central nervous systemare performed at the same time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D. These figures illustrate inclusion levels of exon 20.

FIGS. 2A-F. FIGS. 2A, 2B, 2D, and 2F illustrate the inclusionpercentages of IKBKAP exon 20 in response to different doses ofantisense oligonucleotide compounds. FIGS. 2C and 2F illustrateinclusion percentages of IKBKAP exon 20 in different tissues from ICV orsubcutaneous injections.

FIGS. 3A-B. These figures illustrate minigene constructs.

FIGS. 4A-D. These figures illustrate microwalks of antisenseoligonucleotide compounds on different regions of an IKBKAP gene.

FIG. 5. This figure illustrates the stability of skipped mRNAs with orwithout the premature termination codon using RT-PCR.

DETAILED DESCRIPTION

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “IKBKAP Transcript” means a transcript transcribed froman IKBKAP Gene.

As used herein, “IKBKAP Gene” means GENBANK Accession No NT 008470.16truncated from nucleotides 13290828 to 13358424, designated herein asSEQ ID NO: 1.

As used herein, “aberrant splice site” means a splice site that resultsfrom a mutation in the native DNA and mRNA. In certain embodiments,aberrant splice sites result in mRNA transcripts comprised of adifferent combination of exons. In certain embodiments, aberrant splicesites result in mRNA transcripts with deletions of exons. In certainembodiments, aberrant splice sites result in mRNA transcripts withdeletions of portions of exons, or with extensions of exons, or with newexons. In certain embodiments, aberrant splice sites result in mRNAtranscripts comprising premature stop codons.

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Inreference to an oligonucleotide, chemical modification does not includedifferences only in nucleobase sequence. Chemical modifications ofoligonucleotides include nucleoside modifications (including sugarmoiety modifications and nucleobase modifications) and internucleosidelinkage modifications.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugarmoiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl comprisingat least one substituent group that differs from that of a naturallyoccurring sugar moiety. Substituted sugar moieties include, but are notlimited to furanosyls comprising substituents at the 2′-position, the3′-position, the 5′-position and/or the 4′-position.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside is capable of (1) incorporation into an oligonucleotide and(2) hybridization to a complementary nucleoside. Such structures includerings comprising a different number of atoms than furanosyl (e.g., 4, 6,or 7-membered rings); replacement of the oxygen of a furanosyl with anon-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change inthe number of atoms and a replacement of the oxygen. Such structures mayalso comprise substitutions corresponding to those described forsubstituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugarsurrogates optionally comprising additional substituents). Sugarsurrogates also include more complex sugar replacements (e.g., thenon-ring systems of peptide nucleic acid). Sugar surrogates includewithout limitation morpholino, modified morpholinos, cyclohexenyls andcyclohexitols.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e., noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified.

As used herein, “heterocyclic base” or “heterocyclic nucleobase” means anucleobase comprising a heterocyclic structure.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide.

As used herein, “terminal group” means one or more atoms attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” means an atom or group of atoms bound to anoligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid.

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measureable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenylation, addition of 5′-cap, mRNA turnover), and translation andpost-translational modification.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound hybridizes.

As used herein, “mRNA” means an RNA molecule that encodes a protein.

As used herein, “pre-mRNA” means an RNA transcript that has not beenfully processed into mRNA. Pre-RNA includes one or more introns.

As used herein, “transcript” means an RNA molecule transcribed from DNA.Transcripts include, but are not limited to mRNA, pre-mRNA, andpartially processed RNA.

As used herein, “targeting” or “targeted to” means the association of anantisense compound to a particular target nucleic acid molecule or aparticular region of a target nucleic acid molecule. An antisensecompound targets a target nucleic acid if it is sufficientlycomplementary to the target nucleic acid to allow hybridization underphysiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity under stringent conditions. Complementary oligomericcompounds need not have nucleobase complementarity at each nucleoside.Rather, some mismatches are tolerated. In certain embodiments,complementary oligomeric compounds or regions are complementary at 70%of the nucleobases (70% complementary). In certain embodiments,complementary oligomeric compounds or regions are 80% complementary. Incertain embodiments, complementary oligomeric compounds or regions are90% complementary. In certain embodiments, complementary oligomericcompounds or regions are 95% complementary. In certain embodiments,complementary oligomeric compounds or regions are 100% complementary.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an antisense oligonucleotide specifically hybridizes tomore than one target site.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “motif” means a pattern of chemical modifications in anoligomeric compound or a region thereof. Motifs may be defined bymodifications at certain nucleosides and/or at certain linking groups ofan oligomeric compound.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligomeric compound or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligomeric compound or region thereof. The nucleosides of such anoligomeric compound may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleosides have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substituent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentinvention have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking group,such as an alkyl or hydrocarbyl group, to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms differs from the atomor group of atoms normally present in the named functional group. Incertain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))—(R_(cc))), imino(═NR_(bb)), amido (—C(O)N(R_(bb))(R_(cc)) or—N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(aa))), thioureido(—N(R_(bb))C(S)N(R_(bb))(R_(aa))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(ca)) or —N(R_(bb))S(O)₂R_(bb)). Whereineach R_(aa), R_(bb) and R_(cc) is, independently, H, an optionallylinked chemical functional group or a further substituent group with apreferred list including without limitation, alkyl, alkenyl, alkynyl,aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,heterocyclic and heteroarylalkyl. Selected substituents within thecompounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,ten-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or polycyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

Oligomeric Compounds

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, such oligomeric compounds compriseoligonucleotides optionally comprising one or more conjugate and/orterminal groups. In certain embodiments, an oligomeric compound consistsof an oligonucleotide. In certain embodiments, oligonucleotides compriseone or more chemical modifications. Such chemical modifications includemodifications to one or more nucleoside (including modifications to thesugar moiety and/or the nucleobase) and/or modifications to one or moreinternucleoside linkage.

Certain Sugar Moieties

In certain embodiments, oligomeric compounds of the invention compriseone or more modified nucleosides comprising a modified sugar moiety.Such oligomeric compounds comprising one or more sugar-modifiednucleosides may have desirable properties, such as enhanced nucleasestability or increased binding affinity with a target nucleic acidrelative to oligomeric compounds comprising only nucleosides comprisingnaturally occurring sugar moieties. In certain embodiments, modifiedsugar moieties are substitued sugar moieties. In certain embodiments,modified sugar moieties are bicyclic or tricyclic sugar moieties. Incertain embodiments, modified sugar moieties are sugar surrogates. Suchsugar surogates may comprise one or more substitutions corresponding tothose of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more substituent, including but not limitedto substituents at the 2′ and/or 5′ positions. Examples of sugarsubstituents suitable for the 2′-position, include, but are not limitedto: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE”). Incertain embodiments, sugar substituents at the 2′ position is selectedfrom allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, O—C₁-C₁₀substituted alkyl; O—C₁-C₁₀ alkoxy; O—C₁-C₁₀ substituted alkoxy, OCF₃,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where eachRm and Rn is, independently, H or substituted or unsubstituted C₁-C₁₀alkyl. Examples of sugar substituents at the 5′-position, include, butare not limited to:, 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. Incertain embodiments, substituted sugars comprise more than onenon-bridging sugar substituent, for example, 2′-F-5′-methyl sugarmoieties (see, e.g., PCT International Application WO 2008/101157, foradditional 5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, O—C₁-C₁₀ alkoxy; O—C₁-C₁₀ substituted alkoxy, SH, CN, OCN,CF₃, OCF₃, O-alkyl, S-alkyl, N(R_(m))-alkyl; O— alkenyl, S— alkenyl, orN(R_(m))-alkenyl; O— alkynyl, 5-alkynyl, N(R_(m))-alkynyl;O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl,O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)),where each R_(m) and R_(n) is, independently, H, an amino protectinggroup or substituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituentgroups can be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH—CH₂, O—CH₂—CH—CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂—O—(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—;4′-CH₂-2′,4′-(CH₂)₂-2′,4′-(CH₂)₃-2′,4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, andanalogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15,2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g.,WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, e.g., WO2008/150729, published Dec. 11, 2008);4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004);4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see PCT International ApplicationWO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n), —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—,—C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. USA., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett.,1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007);Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braaschet al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol.Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748,6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S.Patent Publication Nos. US2004/0171570, US2007/0287831, andUS2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574,61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and61/099,844; and PCT International Applications Nos. PCT/US2008/064591,PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surogates comprise a 4′-sulfur atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran. Such tetrahydropyrans may befurther modified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group;

q1, q2, q3, q4, qs, q6 and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q_(b) q₂, q₃, q₄, q₅, q₆ andq₇ is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systemsare known in the art that can be used to modify nucleosides (see, e.g.,review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002,10, 841-854).

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following structure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′,2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

Certain Nucleobases

In certain embodiments, nucleosides of the present invention compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present invention comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyl-adenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

Certain Internucleoside Linkages

In certain embodiments, the present invention provides oligomericcompounds comprising linked nucleosides. In such embodiments,nucleosides may be linked together using any internucleoside linkage.The two main classes of internucleoside linking groups are defined bythe presence or absence of a phosphorus atom. Representative phosphoruscontaining internucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of the oligomericcompound. In certain embodiments, internucleoside linkages having achiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Certain Motifs

In certain embodiments, the present invention provides oligomericcompounds comprising oligonucleotides. In certain embodiments, sucholigonucleotides comprise one or more chemical modification. In certainembodiments, chemically modified oligonucleotides comprise one or moremodified nucleosides. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleosides comprisingmodified sugars. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleosides comprisingone or more modified nucleobases. In certain embodiments, chemicallymodified oligonucleotides comprise one or more modified internucleosidelinkages. In certain embodiments, the chemical modifications (sugarmodifications, nucleobase modifications, and/or linkage modifications)define a pattern or motif. In certain embodiments, the patterns ofchemical modifications of sugar moieties, internucleoside linkages, andnucleobases are each independent of one another. Thus, anoligonucleotide may be described by its sugar modification motif,internucleoside linkage motif and/or nucleobase modification motif (asused herein, nucleobase modification motif describes the chemicalmodifications to the nucleobases independent of the sequence ofnucleobases).

Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar modification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar modification motif, which comprises twoexternal regions or “wings” and an internal region or “gap.” The threeregions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar modification motifs of the two wings are the sameas one another (symmetric gapmer). In certain embodiments, the sugarmodification motifs of the 5′-wing differs from the sugar modificationmotif of the 3′-wing (asymmetric gapmer). In certain embodiments,oligonucleotides comprise 2′-MOE modified nucleosides in the wings and2′-F modified nucleosides in the gap.

In certain embodiments, oligonucleotides are fully modified. In certainsuch embodiments, oligonucleotides are uniformly modified. In certainembodiments, oligonucleotides are uniform 2′-MOE. In certainembodiments, oligonucleotides are uniform 2′-F. In certain embodiments,oligonucleotides are uniform morpholino. In certain embodiments,oligonucleotides are uniform BNA. In certain embodiments,oligonucleotides are uniform LNA. In certain embodiments,oligonucleotides are uniform cEt.

In certain embodiments, oligonucleotides comprise a uniformly modifiedregion and additional nucleosides that are unmodified or differentlymodified. In certain embodiments, the uniformly modified region is atleast 5, 10, 15, or 20 nucleosides in length. In certain embodiments,the uniform region is a 2′-MOE region. In certain embodiments, theuniform region is a 2′-F region. In certain embodiments, the uniformregion is a morpholino region. In certain embodiments, the uniformregion is a BNA region. In certain embodiments, the uniform region is aLNA region. In certain embodiments, the uniform region is a cEt region.

In certain embodiments, the oligonucleotide does not comprise more than4 contiguous unmodified 2′-deoxynucleosides. In certain circumstances,antisense oligonucleotides comprising more than 4 contiguous2′-deoxynucleosides activate RNase H, resulting in cleavage of thetarget RNA. In certain embodiments, such cleavage is avoided by nothaving more than 4 contiguous 2′-deoxynucleosides, for example, wherealteration of splicing and not cleavage of a target RNA is desired.

Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif, as described above for sugar modification motif. In suchembodiments, the internucleoside linkages in each of two wing regionsare different from the internucleoside linkages in the gap region. Incertain embodiments, the internucleoside linkages in the wings arephosphodiester and the internucleoside linkages in the gap arephosphorothioate. The sugar modification motif is independentlyselected, so such oligonucleotides having a gapped internucleosidelinkage motif may or may not have a gapped sugar modification motif andif it does have a gapped sugar motif, the wing and gap lengths may ormay not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present invention comprise a region of uniformlymodified internucleoside linkages. In certain such embodiments, theoligonucleotide comprises a region that is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide is uniformly linked by phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate and at least oneinternucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certain suchembodiments, nucleobase modifications are arranged in a gapped motif. Incertain embodiments, nucleobase modifications are arranged in analternating motif. In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

Certain Overall Lengths

In certain embodiments, the present invention provides oligomericcompounds including oligonucleotides of any of a variety of ranges oflengths. In certain embodiments, the invention provides oligomericcompounds or oligonucleotides consisting of X to Y linked nucleosides,where X represents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X<Y. For example, in certainembodiments, the invention provides oligomeric compounds which compriseoligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linkednucleosides. In embodiments where the number of nucleosides of anoligomeric compound or oligonucleotide is limited, whether to a range orto a specific number, the oligomeric compound or oligonucleotide may,nonetheless further comprise additional other substituents. For example,an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotideshaving 31 nucleosides, but, unless otherwise indicated, such anoligonucleotide may further comprise, for example one or moreconjugates, terminal groups, or other substituents. In certainembodiments, a gapmer oligonucleotide has any of the above lengths.

One of skill in the art will appreciate that certain lengths may not bepossible for certain motifs. For example: a gapmer having a 5′-wingregion consisting of four nucleotides, a gap consisting of at least sixnucleotides, and a 3′-wing region consisting of three nucleotides cannothave an overall length less than 13 nucleotides. Thus, one wouldunderstand that the lower length limit is 13 and that the limit of 10 in“10-20” has no effect in that embodiment.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range. For example, an oligonucleotide consisting of20-25 linked nucleosides comprising a 5′-wing consisting of 5 linkednucleosides; a 3′-wing consisting of 5 linked nucleosides and a centralgap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap(before reaching the overall length limitation of 25). Such additionalnucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing.

Certain Oligonucleotides

In certain embodiments, oligonucleotides of the present invention arecharacterized by their sugar motif, internucleoside linkage motif,nucleobase modification motif and overall length. In certainembodiments, such parameters are each independent of one another. Thus,each internucleoside linkage of an oligonucleotide having a gapmer sugarmotif may be modified or unmodified and may or may not follow the gapmermodification pattern of the sugar modifications. Thus, theinternucleoside linkages within the wing regions of a sugar-gapmer maybe the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region. Likewise,such sugar-gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.Herein if a description of an oligonucleotide or oligomeric compound issilent with respect to one or more parameter, such parameter is notlimited. Thus, an oligomeric compound described only as having a gapmersugar motif without further description may have any length,internucleoside linkage motif, and nucleobase modification motif. Unlessotherwise indicated, all chemical modifications are independent ofnucleobase sequence.

Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamics, pharmacokinetics, stability, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligomericcompound, such as an oligonucleotide. Conjugate groups includes withoutlimitation, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groupshave been described previously, for example: cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucleic Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-β-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucleic Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

In certain embodiments, conjugate groups are directly attached tooligonucleotides in oligomeric compounds. In certain embodiments,conjugate groups are attached to oligonucleotides by a conjugate linkinggroup. In certain such embodiments, conjugate linking groups, including,but not limited to, bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Conjugate linkinggroups are useful for attachment of conjugate groups, such as chemicalstabilizing groups, functional groups, reporter groups and other groupsto selective sites in a parent compound such as for example anoligomeric compound. In general a bifunctional linking moiety comprisesa hydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such asa chemical functional group or a conjugate group. In some embodiments,the conjugate linker comprises a chain structure or an oligomer ofrepeating units such as ethylene glycol or amino acid units. Examples offunctional groups that are routinely used in a bifunctional linkingmoiety include, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties includepyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′ end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group.

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, oligomeric compounds comprise anoligonucleotide. In certain embodiments, an oligomeric compoundcomprises an oligonucleotide and one or more conjugate and/or terminalgroups. Such conjugate and/or terminal groups may be added tooligonucleotides having any of the chemical motifs discussed above.Thus, for example, an oligomeric compound comprising an oligonucleotidehaving region of alternating nucleosides may comprise a terminal group.

Antisense Compounds

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. Such antisense compounds are capable ofhybridizing to a target nucleic acid, resulting in at least oneantisense activity. In certain embodiments, antisense compoundsspecifically hybridize to one or more target nucleic acid. In certainembodiments, a specifically hybridizing antisense compound has anucleobase sequence comprising a region having sufficientcomplementarity to a target nucleic acid to allow hybridization andresult in antisense activity and insufficient complementarity to anynon-target so as to avoid non-specific hybridization to any non-targetnucleic acid sequences under conditions in which specific hybridizationis desired (e.g., under physiological conditions for in vivo ortherapeutic uses, and under conditions in which assays are performed inthe case of in vitro assays).

In certain embodiments, the present invention provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

Certain Target Nucleic Acids and Mechanisms

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid. In certain embodiments, the target nucleic acid is anendogenous RNA molecule. In certain embodiments, the target nucleic acidis a pre-mRNA. In certain embodiments, the target nucleic acid is anIKBKAP transcript. In certain embodiments, the target RNA is an IKBKAPpre-mRNA.

In certain embodiments, an antisense compound is complementary to aregion of an IKBKAP pre-mRNA. In certain embodiments, an antisensecompound is complementary within a region of an IKBKAP pre-mRNAcomprising intron 19, intron 20, or exon 20. In certain embodiments, anantisense compound is complementary to a region of an IKBKAP pre-mRNAconsisting of intron 19, intron 20, or exon 20. In certain embodiments,an antisense compound is complementary to a region of an IKBKAP pre-mRNAconsisting of exon 20 or intron 20. In certain embodiments, an antisensecompound is complementary to a region of an IKBKAP pre-mRNA withinintron 19. In certain embodiments, an antisense compound iscomplementary to a region of an IKBKAP pre-mRNA within intron 20. Incertain embodiments, an antisense compound is complementary to a regionof an IKBKAP pre-mRNA within exon 20.

In certain embodiments, an antisense oligonucleotide modulates splicingof a pre-mRNA. In certain embodiments, an antisense oligonucleotidemodulates splicing an IKBKAP pre-mRNA. In certain such embodiments, theIKBKAP pre-mRNA is transcribed from a mutant variant of IKBKAP. Incertain embodiments, the mutant variant comprises an aberrant splicesite. In certain embodiments, the aberrant splice site of the mutantvariant comprises a mutation that weakens the 5′-splice site of exon 20.In certain embodiments, an antisense oligonucleotide reduces aberrantsplicing of an IKBKAP pre-mRNA. In certain embodiments, an antisenseoligonucleotide increases the amount of exon 20 included in normallyspliced IKBKAP mRNA. In certain embodiments, an antisenseoligonucleotide increases the amount of exon 20 skipped IKBKAP mRNA.

Certain Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active antisense oligomericcompound.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present invention provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human.

In certain embodiments, the present invention provides methods ofadministering a pharmaceutical composition comprising an oligomericcompound of the present invention to an animal. Suitable administrationroutes include, but are not limited to, oral, rectal, transmucosal,intestinal, enteral, topical, suppository, through inhalation,intrathecal, intracerebroventricular, intraperitoneal, intranasal,intraocular, intratumoral, and parenteral (e.g., intravenous,intramuscular, intramedullary, and subcutaneous). In certainembodiments, pharmaceutical intrathecals are administered to achievelocal rather than systemic exposures. For example, pharmaceuticalcompositions may be injected directly in the area of desired effect(e.g., into the eyes, ears).

In certain embodiments, a pharmaceutical composition is administered toan animal having at least one symptom associated with FamilialDysautonomia. In certain embodiments, such administration results inamelioration of at least one symptom. In certain embodiments,administration of a pharmaceutical composition to an animal results in adecrease of aberrantly spliced IKBKAP mRNA in a cell of the animal. Incertain embodiments, such administration results in an increase innormally spliced IKBKAP mRNA and/or an increase in mRNA containing exon20. In certain embodiments, such administration results in an increasein normally spliced IKBKAP mRNA and/or an increase in mRNA containingexons 20-37. In certain embodiments, such administration results in anincrease in normally spliced IKBKAP mRNA and/or a decrease in exon 20skipped mRNA. In certain embodiments, such administration results in adecrease in truncated IKAP protein and an increase in normal IKAPprotein. In certain embodiments, administration of a pharmacueuticalcomposition results in amelioration of: anhidrosis, decreased taste,depressed deep tendon reflexes, postural hypertension, loss of pain andtemperature perception, alacrima, gastroesophageal reflux, andscoliosis. In certain embodiments, such amelioration is the reduction inseverity of such defects. In certain embodiments, amelioration is thedelayed onset of such defects. In certain embodiments, amelioration isthe slowed progression of such defects. In certain embodiments,amelioration is the prevention of such defects. In certain embodiments,amelioration is the slowed progression of such defects. In certainembodiments, amelioration is the reversal of such defects.

In certain embodiments, one tests an animal for defects in the IKBKAPgene. In certain embodiments, one identifies an animal having one ormore splicing defects in the IKBKAP gene. In certain embodiments, apharmaceutical composition is administered to an animal identified ashaving a defect in the IKBKAP gene. In certain embodiments, the animalis tested following administration.

In certain embodiments, one tests for defects in a human IKBKAPtransgene. In certain embodiments, one identifies an animal having oneor more splicing defects in a human IKBKAP transgene. In certainembodiments, a pharmaceutical composition is administered to an animalidentified as having a defect in a human IKBKAP transgene. In certainembodiments, the animal is tested following administration.

In certain embodiments, one tests an animal for defects in a mouseIkbkap gene. In certain embodiments, one identifies an animal having oneor more splicing defects in a mouse Ikbkap gene. In certain embodiments,a pharmaceutical composition is administered to an animal identified ashaving a defect in the IKBKAP gene. In certain embodiments, the animalis tested following administration.

The disclosure also provides an antisense compound as described herein,for use in any of the methods as described herein. For example, theinvention provides an antisense compound comprising an antisenseoligonucleotide for use in treating a disease or condition associated FDby administering the antisense compound directly into the centralnervous system (CNS) or cerebrospinal fluid (CSF).

In certain embodiments, the antisense compound is administeredsystemically. In certain embodiments, the systemic administration is byintravenous or intraperitoneal injection. In certain embodiments,systemic administration and the administration into the central nervoussystem are performed at the same time. In certain embodiments, systemicadministration and the administration into the central nervous systemare performed at different times.

In certain embodiments, the invention provides systemic administrationof antisense compounds, either alone or in combination with deliveryinto the CSF. In certain embodiments, pharmaceutical compositions areadministered systemically. In certain embodiments, pharmaceuticalcompositions are administered subcutaneously. In certain embodiments,pharmaceutical compositions are administered intravenously. In certainembodiments, pharmaceutical compositions are administered byintramuscular injection.

In certain embodiments, pharmaceutical compositions are administeredboth directly to the CSF (e.g., IT and/or ICV injection and/or infusion)and systemically.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligomeric compound having the nucleobase sequence “ATCGATCG”encompasses any oligomeric compounds having such nucleobase sequence,whether modified or unmodified, including, but not limited to, suchcompounds comprising RNA bases, such as those having sequence “AUCGAUCG”and those having some DNA bases and some RNA bases such as “AUCGATCG”and oligomeric compounds having other modified or naturally occurringbases, such as “AT^(me)CGAUCG,” wherein ^(me)C indicates a cytosine basecomprising a methyl group at the 5-position.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1 Construction of Minigenes Containing Genomic Fragments of theInhibitor-Kappa B Kinase Associated Protein (IKBKAP) Gene

Familial dysautonomia (FD) is caused by a point mutation at the 5′splice site of intron 20, leading to aberrant splicing and the skippingof exon 20 of the IKBKAP genomic sequence (Anderson, S. L. et al., 2001.Am J Hum Genet. 68:753-758). Hence, IKBKAP minigenes were constructed bycloning the genomic fragments comprising either exon 19 to exon 21(designated herein as wt19-21) or exon 19 to exon 22 (designated hereinas wt19-22).

The IKBKAP genomic fragments spanning exons 19-21 and 19-22 wereamplified using specific primers. The genomic fragment for wt19-21 wasamplified using the forward primer sequence IKAP19F6(GGGGAAGGATCCGCCATGGAGTTAATGGTGTGTTTAGCATTACAGG, designated herein asSEQ ID NO: 2) and reverse primer sequence IKAP21R3(GGGGAATCTAGACTTAGGGTTATG ATCATAAATCAGATTGAG, designated herein as SEQID NO: 3). The genomic fragment for wt19-22 was amplified using theforward primer sequence IKAP19F6 and reverse primer sequence IKAP22R(GGGGAATCTAGATTACTTCAATTCTGTAAAAAACAAGTTAATATG, designated herein as SEQID NO: 4). The IKBKAP gene in human genomic DNA (Promega) was used as atemplate. The major mutation found in FD (IVS20+6T→C) (Dong, J. et al.,2002. Am. J. Med. Genet. 110: 253-257) was introduced into both thewt19-21 and wtl 9-22 minigenes by site-directed mutagenesis to createthe minigenes mt19-21 and mtl 9-22. All four minigene fragments wereindividually cloned into the mammalian expression vector, pcDNA3.1(Invitrogen). An in-frame ATG as a first codon within a Kozak consensussequence at the 5′ end, downstream of the cytomegalovirus promoter, aswell as a stop codon at the 3′ end, upstream of a poly(A) signal fromthe pcDNA3.1 vector were also introduced (FIG. 4).

Each minigene vector construct was transfected individually into HEK-293cells cultured in Dulbecco's modified Eagle's medium (Invitrogen)supplemented with 10% (v/v) fetal bovine serum. The transfection wasconducted by electroporation (Gene Pulsar II apparatus, Bio-Rad) toco-transfect 3 μg of the construct into 7×10⁵ HEK-293 cells resuspendedin 70 μL volume of Optimem (Invitrogen) and plated in 6-well plates, asdescribed previously (Hua, Y., et al., 2007. PLoS Biol 5:e73). After 72hrs, cDNA synthesized from total RNA extracted from HEK-293 cells wasamplified, as described previously (Hua, Y., et al., 2007. PLoS Biol5:e73). wt19-21 and mt19-21 were amplified with forward primer pcDNAF(TAATACGACTCACTATAGGG, designated herein as SEQ ID NO: 5) and reverseprimer IKAP21R4 (CTTAGGGTTATGATCATAAATCAG, designated herein as SEQ IDNO: 6). wt19-22 and mt-19-22 were amplified with forward primer pcDNAFand reverse primer IKAP22R2 (TTCAATTCTGTAAAAAACAAG, designated herein asSEQ ID NO: 7). Consistent predominant skipping of exon 20 was observedin the mutant versions of the minigenes, thus recapitulating theaberrant splicing observed in FD patients.

Example 2 Effect of Antisense Oligonucleotides on Exon 20 Skipping inthe IKBKAP Minigenes

Antisense oligonucleotides were designed targeting a human IKBKAPnucleic acid and were tested for their effects on IKBKAP pre-mRNA invitro. Together, the overlapping antisense oligonucleotides spanned theentire 74-nucleotide region of the IKBKAP exon 20 sequence, as well asthe 100-nucleotide intronic regions immediately upstream and downstreamof exon 20. The antisense oligonucleotides are presented in Table 1, andwere designed as uniform 2′-O-methoxyethyl ribose (MOE) oligonucleotideswith phosphate backbones. Each oligonucleotide is 15 nucleosides inlength. All cytosine residues throughout the oligonucleotide are5-methylcytosines. ‘Start Site’ indicates the 5′-most nucleoside towhich the oligonucleotide is targeted in the human gene sequence. “Stopsite” indicates the 3′-most nucleoside to which the oligonucleotide istargeted in the human gene sequence. Each oligonucleotide listed inTable 1 is targeted to the human IKBKAP genomic sequence (the complementof GENBANK Accession No NT 008470.16 truncated from nucleotides 13290828to 13358424, designated herein as SEQ ID NO: 1). ISIS 414161 has onemismatch with SEQ ID NO: 1.

Cultured HEK-293 cells harboring the mtl 9-21 minigene vector constructat a density of 7×10⁵ cells per well were transfected usingelectroporation (Gene Pulsar II apparatus, Bio-Rad) with 0.007 nmolantisense oligonucleotide, as previously described (Hua, Y., et al.,2007. PLoS Biol 5:e73). ISIS 383548, ISIS 383553, and ISIS 383874, whichwere used as control oligonucleotides that do not cause any exonskipping, were similarly transfected. These oligonucleotides served ascontrols for non-specific effects of uniform MOE oligonucleotides withphosphate backbones. Cells harboring the wt19-21 minigene and themt19-21 minigene alone were also cultured and were used as controls forexon 20 inclusion levels. Two days later, cDNA synthesized from totalRNA extracted from HEK-293 cells was amplified using the forward primerpcDNAF and reverse primer sequence IKAP21R4 to assay the splicingpattern of expressed RNAs by RT-PCR. To calculate exon 20 inclusionlevels, the PCR amplicons were labeled with a³²P-dCTP. The PCR productswere then separated by native PAGE, followed by phosphorimage analysison a FUJIFILM FLA-5100 instrument (Fuji Medical Systems USA Inc.). Theband intensities were quantified using Multi Gauge software Version 2.3(FUJIFILM), and values were normalized for the G+C content according tothe DNA sequence.

The results are presented in Table 1 and FIG. 4. The results indicatethat 6 consecutive antisense oligonucleotides, ISIS 414161, ISIS 414162,ISIS 414163, ISIS 414164, ISIS 414165, and ISIS 414166, targeting a40-nucleotide intronic region immediately downstream of the 5′ splicesite of exon 20 markedly increased inclusion of exon 20. This suggeststhe presence of multiple splicing silencer elements or inhibitorysecondary structures within this region, designated herein as ISS-40.Three more antisense oligonucleotides, ISIS 414135, ISIS 414136, andISIS 414137, which target a 20-nucleotide region in the upstream intron19 (designated herein as ISS-20) also had a positive effect on exon 20inclusion (Table 1 and FIG. 4). Antisense oligonucleotides targetingexon 20 resulted in near-complete exon skipping. Treatment withantisense oligonucleotides targeting the 3′ and 5′ splice sites causedincreased skipping of exon 20. Certain other antisense oligonucleotidestargeting intronic regions also caused increased skipping because theytargeted important cis-acting splicing elements, the polypyrimidinetract, or the 5′ splice site of intron 20. ISIS 414167 and ISIS 414168,which target an intronic splicing enhancer (designated herein asISE-20), also significantly decreased the levels of the included RNAisoform compared to the untreated control. The results from the threesets of control oligonucleotide-treated cells were combined and theaverage is presented in Table 1, designated as ‘controloligonucleotide’. ‘n/a’ indicates ‘not applicable. ‘n.d.’ indicates thatthere is no data for that particular oligonucleotide.

TABLE 1 Uniform MOE antisense oligonucleotides targetingintrons 19 and 20 and exon 20 of SEQ ID NO: 1 Start Stop Target % SEQConstruct ISIS No Sequence Site Site Region inclusion ID NO mt19-21414129 AGAGAATTACCACAA 34622 34636 intron 19 23 8 414130 TTCACAGAGAATTAC34627 34641 intron 19 25 9 414131 AACTCTTCACAGAGA 34632 34646 intron 1930 10 414132 TACCTAACTCTTCAC 34637 34651 intron 19 19 11 414133CATTTTACCTAACTC 34642 34656  intron 19 25 12 414134 TACACCATTTTACCT34647 34661  intron 19 25 13 414135 CAGGATACACCATTT 34652 34666intron 19 46 14 414136 ATAGCCAGGATACAC 34657 34671 intron 19 45 15414137 TTTAAATAGCCAGGA 34662 34676 intron 19 52 16 414138AAACATTTAAATAGC 34667 34681 intron 19 23 17 414139 GTAGAAAACATTTAA 3467234686 intron 19 20 18 414140 ATTAAGTAGAAAACA 34677 34691 intron 19 13 19414141 TITTAATTAAGTAGA 34682 34696 intron 19 24 20 414142AACATTTTTAATTAA 34687 34701 intron 19 23 21 414143 GCAGTAACATTTTTA34692  34706  intron 19 8 22 414144 TTAAAGCAGTAACAT 34697  34711  intron 19 7 23 414145 ATAAATTAAAGCAGT 34702 34716 intron 19 3 24 414146CTTAAATAAATTAAA 34707  34721 intron 19 26 25 414147 GTTTCCCCTTGGCAT34722  34736  exon 20 3 26 414148 TCTAAGTTTCCCCTT 34727  34741  exon 207 27 414149 CAACTTCTAAGTTTC 34732  34746  exon 20 20 28 414150ATGAACAACTTCTAA 34737  34751  exon 20 5 29 414151 CGATGATGAACAACT 34742 34756 exon 20 0 30 414152 GGGCTCGATGATGAA 34747  34761  exon 20 3 31414153 AACCAGGGCTCGATG 34752  34766 exon 20 5 32 414154 GCTAAAACCAGGGCT34757  34771  exon 20 n.d. 33 414155 TCTGAGCTAAAACCA 34762  34776 exon 20 6 34 414156 CCGAATCTGAGCTAA 34767  34781  exon 20 5 35 414157CACTTCCGAATCTGA 34772  34786  exon 20 15 36 414158 CCAACCACTTCCGAA34777  34791  exon 20 1 37 414159 TTGTCCAACCACTTC 34781  34795  exon 202 38 414160 TACAATGGCGCTTAC 34796  34810 intron 20 14 39 414161AACAGTACAATGGCG 34801  34815 intron 20 88 40 414162 TCGCAAACAGTACAA34806  34820  intron 20 79 41 414163 ACTAGTCGCAAACAG 34811 34825intron 20 74 42 414164 AGCTAACTAGTCGCA 34816 34830 intron 20 78 43414165 TCACAAGCTAACTAG 34821 34835 intron 20 66 44 414166ATAAATCACAAGCTA 34826 34840 intron 20 40 45 414167 CACACATAAATCACA 3483134845 intron 20 13 46 414168 GTCTTCACACATAAA 34836 34850 intron 20 17 47414169 TTATTGTCTTCACAC 34841 34855 intron 20 21 48 414170AATACTTATTGTCTT 34846 34860 intron 20 32 49 414171 AATAAAATACTTATT 3485134865 intron 20 32 50 414172 ATTGTAATAAAATAC 34856 34870 intron 20 31 51414173 TCGAAATTGTAATAA 34861 34875 intron 20 31 52 414174AGTTCTCGAAATTGT 34866 34880 intron 20 29 53 414175 TTTTAAGTTCTCGAA 3487134885 intron 20 27 54 414176 CATAATTTTAAGTTC 34876 34890 intron 20 31 55414177 CTTTTCATAATTTTA 34881 34895 intron 20 25 56 wt19-21 n/a n/a n/an/a n/a 99 Untreated  n/a n/a n/a n/a n/a 29 mt19-21 mt19-21 Control383548 TTTATATGGATGTTA n/a n/a n/a 24 57 oligos AAAAG 383553 AAAAGCATTTTGTTT 58 CACAA 383874 ATTTTGTCTGAAACC 59

Skipping of exon 20 causes a frameshift that introduces a prematuretermination codon (PTC) in exon 21, thereby making the mRNA potentiallysusceptible to degradation according to the characterized rules of thenonsense-mediated mRNA decay (NMD) pathway (Nagy, E., and Maquat, L. E.1998. Trends Biochem Sci 23:198-199). A similar experiment to the onedescribed above was conducted utilizing the wt19-22 and the mt19-22minigenes to determine if the NMD pathway controls the stability of theskipped mRNA isoform. The same pattern of inclusion or skipping of exon20 was observed with the wt19-22 and mtl 9-22 minigenes as observed withthe corresponding 19-21 minigenes. Therefore, there is no evidence thatthe skipped mRNA isoform resulting from mt19-22 minigene was subject toNMD.

To confirm this finding, a single nucleotide in exon 21 of the mt19-22minigene was deleted to restore the reading frame and remove thepremature termination codon (PTC). This minigene was designated asmt19-22FC minigene (FIG. 3). The three minigenes, wt19-22, mt19-22 andmt19-22FC, were individually transfected into HEK-293 cells using thesame protocol as described above. The expressed RNA was analyzed byRT-PCR. Consistent with the observation made in the study with antisenseoligonucleotide transfection described above, the skipped mRNAs with orwithout the PTC were equally stable (FIG. 5). This confirms that atleast in HEK-293 cells, the skipped mRNA isoform is not subject to NMD.

Example 3 Effect of Oligonucleotides Designed by Microwalk on ExonSkipping in the IKBKAP Minigenes

Additional oligonucleotides were designed targeting the first30-nucleotide stretch of ISS-40. These oligonucleotides were designed bychoosing sequences shifted in one nucleotide increments upstream anddownstream (i.e., a “microwalk”) of ISS-40, starting from the +6position in exon 20. The antisense oligonucleotides are presented inTable 2 and FIG. 4D, and were designed as uniform 2′-β-methoxyethylribose (MOE) oligonucleotides with phosphate backbones. Eacholigonucleotide is 20 nucleosides in length. All cytosine residuesthroughout the oligonucleotide are 5-methylcytosines. ‘Start Site’indicates the 5′-most nucleoside to which the oligonucleotide istargeted in the human gene sequence. “Stop site” indicates the 3′-mostnucleoside to which the oligonucleotide is targeted in the human genesequence. Each oligonucleotide listed in Table 2 is targeted to intron20 of the human IKBKAP genomic sequence (the complement of GENBANKAccession No NT 008470.16 truncated from nucleotides 13290828 to13358424, designated herein as SEQ ID NO: 1). These oligonucleotideswere tested in vitro. ISIS 414161, ISIS 414162, ISIS 414163, ISIS414163, ISIS 414164, ISIS 414165, and ISIS 414166, which showed a highpercentage of inclusion, were also included in the assay.

Cultured HEK-293 cells harboring the mt19-21 minigene vector constructat a density of 7×10⁵ cells per well were transfected usingelectroporation (Gene Pulsar II apparatus, Bio-Rad) with 0.007 nmolantisense oligonucleotide, as previously described (Hua, Y., et al.,2007. PLoS Biol 5:e73). Control oligonucleotides that do not cause anyexon skipping were similarly transfected and served as controls fornon-specific effects of uniform MOE oligonucleotides with phosphatebackbones. Cells harboring the wtl 9-21 minigene and the mt19-21minigene alone were also cultured and were used as controls for exon 20inclusion levels. Two days later, cDNA synthesized from total RNAextracted from HEK-293 cells was amplified using the forward primerpcDNAF and reverse primer sequence IKAP21R4 to assay the splicingpattern of expressed RNAs by RT-PCR. To calculate exon 20 inclusionlevels, the PCR amplicons were labeled with α³²P-dCTP. The PCR productswere then separated by native PAGE, followed by phosphorimage analysis.The band intensities were quantified and values were normalized for theG+C content according to the DNA sequence. The results are presented inTable 2. It was observed that treatment of the cells with ISIS 421992restored exon 20 inclusion levels to 96% in the mutant minigene. It wasalso observed that the 20-mer antisense oligonucleotides have a strongerpositive effect on exon 20 splicing than the 15-mer antisenseoligonucleotides targeting the same region.

TABLE 2 Uniform MOE antisense oligonucleotidestargeting intron 20 of SEQ ID NO: 1 Start Stop % SEQ Construct ISIS NoSequence Site Site inclusion ID NO mt19-21 414161 AACAGTACAATGGCG 3480134815 79 40 414162 TCGCAAACAGTACAA 34806 34820 82 41 414163ACTAGTCGCAAACAG 34811 34825 74 42 414164 AGCTAACTAGTCGCA 34816 34830 7443 414165 TCACAAGCTAACTAG 34821 34835 64 44 414166 ATAAATCACAAGCTA 3482634840 38 45 421991 TCGCAAACAGTACAATGGCG 34801 34820 91 60 421992GTCGCAAACAGTACAATGGC 34802 34821 96 61 421993 AGTCGCAAACAGTACAATGG 3480334822 87 62 421994 TAGTCGCAAACAGTACAATG 34804 34823 91 63 421995CTAGTCGCAAACAGTACAAT 34805 34824 92 64 421996 ACTAGTCGCAAACAGTACAA 3480634825 89 65 421997 AACTAGTCGCAAACAGTACA 34807 34826 81 66 421998TAACTAGTCGCAAACAGTAC 34808 34827 81 67 421999 CTAACTAGTCGCAAACAGTA 3480934828 82 68 422000 GCTAACTAGTCGCAAACAGT 34810 34829 63 69 wt19-21 n/an/a n/a n/a 100 n/a Untreated n/a n/a n/a n/a 33 n/a mt19-21 mt19-21Control TTTATATGGATGTTAAAAAG n/a n/a 32 57 oligos AAAAGCATTTTGTTTCACAA58 ATTTTGTCTGAAACC 59

Example 4 Effect of ISIS 421992 in FD-Derived Fibroblasts

To investigate the effect of ISIS 421992 on patient-derived fibroblasts,the patient skin fibroblast line GM04899 (Coriell Cell Repository) wasutilized. The cell line was derived from an individual homozygous forthe major FD mutation.

GM04899 was cultured in minimal essential medium (Invitrogen)supplemented with non-essential amino acids (Invitrogen) and 20% (v/v)fetal bovine serum. The cells were grown to 40-50% confluence in 10-cmdishes. Cells were transfected with 2 nM, 5 nM, 25 nM, or 125 nMconcentrations of ISIS 421992 using 12 μL Lipofectamine 2000transfection reagent (Invitrogen). Two days later, cDNA synthesized fromtotal RNA extracted from HEK-293 cells was amplified using the forwardprimer pcDNAF and reverse primer sequence LKAP21R4 to assay the splicingpattern of expressed RNAs by RT-PCR. To calculate exon 20 inclusionlevels, the PCR amplicons were labeled with a³²P-dCTP. The PCR productswere then separated by native PAGE, followed by phosphorimage analysis.The band intensities were quantified and values were normalized for theG-FC content according to the DNA sequence. The results are presented inTable 3 and FIG. 1. Multiple lanes for each condition representindependent experiments.

Treatment with ISIS 421992 almost completely suppressed the splicingdefect, as demonstrated by the percent inclusion of exon 20 in Table 3and the left panel of FIG. 1A. Kinetin (6-furfurylaminopurine) has beenshown to improve splicing and increase wild-type IKBKAP mRNA and IKAPprotein expression in FD cell lines (Hims, M. M. et al., 2007. J. Mol.Med. 85: 149-161). Treatment with ISIS 421992 was as effective astreatment with kinetin for 3 days in restoring full-length mRNA levels(FIG. 1A, right panel). A batch of cells was treated with solvent (NaOH)only of the kinetin solution as a control. RNA from IMR90, a wild-typenormal diploid lung fibroblast cell line was used as positive control.The results are expressed as percent inclusion of exon 20 compared tothe exon 20 inclusion (100%) of IKBKAP mRNA in IMR90 cells.

Treatment with ISIS 421992 at 5 nM also resulted in a significantincrease in IKAP protein levels, when assayed 3 days after transfection.Protein samples were obtained by Trizol extraction and separated bySDS-PAGE. The bands were transferred onto a nitrocellulose membrane andprobed with an anti-IKAP antibody (abcam # ab56362) (1: 1,000 dilutionin 5% milk in TBST) for 12 hrs. The membrane was washed 5 times withTBST for 5 min each and then probed with secondary antibody 800 nm LiCor(1: 5,000 in 5% milk in TBST) for 1 hr in the dark. The membrane wassubsequently washed 5 times with TBST for 5 min each. The bands werethen exposed at 800 nm and band intensity was quantified using anOdyssey (LiCor) instrument. The results are presented in Table 4 aspercent increase in band intensity compared to the controloligonucleotide-treated bands. The data indicate that treatment withISIS 421992 significantly increased IKAP protein levels compared to thecontrol. Note that under these conditions, kinetin did not increase IKAPprotein levels.

TABLE 3 Exon 20 inclusion after treatment of GM04899 with ISIS 421992 %inclusion Untreated 69 Control oligo treated 67 ISIS 421992 2 nM 79 ISIS421992 5 nM 95 ISIS 421992 25 nM 96 ISIS 421992 125 nM 97solvent-treated 67 kinetin-treated 98

TABLE 4 Percent incease in protein band intensity after treatment ofGM04899 with ISIS 421992 % ISIS 421992 2 nM 76 solvent-treated 10kinetin-treated 11

Example 5 Effect of ISIS 421992 in a Transgenic Mouse Model

To investigate the effect of ISIS 421992 in an animal model, transgenicmice that carry the entire human IKBKAP gene with the major FD mutation,in addition to being homozygous wild type at the mouse Ikbkap locus,were obtained from an NIH core facility. Though the transgenic mice donot show any overt disease phenotype, due to the presence of thewild-type mouse Ikbkap gene, the mRNA expressed by the mutant humanIKBKAP transgene does show a pattern of skipping similar to that of FDpatients (Hims, M. M. et al., 2007. Genomics. 90: 389-396).

Adult mice were treated with ISIS 421992 administered byintracerebroventricular infusion at the rate of 50 μg/day, 100 μg/day,or 200 μg/day. The protocol has been previously described by Hua et al.(Genes Dev. 2010. 24: 1634-1644). Adult mice 3-4 months old and weighing20-30 g, were anesthetized and placed on a digital stereotaxicinstrument (David Kopf Instruments). A small burr hole at the surgicalsite 1.8 mm lateral to the sagittal suture and 0.3 mm posterior to thebregma suture was drilled through the skull above the right lateralventricle. A cannula with a 2.2 mm stylet was positioned in the hole.The cannula was connected to an Alzet micro-osmotic pump (model 1007D,Durect Corporation) with a vinyl catheter. The pump, prefilled with theoligonucleotide solution or PBS only was implanted subcutaneously on theback and continuously infused the solution through the cannula into thelateral ventricle at a rate of 0.5 μL per hour. After a week of ICVinfusion, the mice were euthanized on day 8 and RNA from the thoracicspinal cord of the transgenic mice was extracted using Trizol andfollowing the manufacturer's protocol. Human IKBKAP mRNA levels weremeasured and the results are presented in Table 5 and FIG. 2A. Multiplelanes for each condition represent independent experiments. The dataindicate that there was a dose-dependent increase in the inclusion ofexon 20 in human IKBKAP mRNA levels in these mice.

TABLE 5 Percent inclusion of exon 20 in human IKBKAP mRNA levels intransgenic mice Treatment Dose (μg/day) % PBS — 6 ISIS 421992 50 40 10046 200 60

Neonatal transgenic mice were also treated with ISIS 421992 administeredas a single ICV injection dose of 2.5 μg, 5 μg, 10 μg, 20 μg, or 30 μg.A group of neonatal mice were treated with ISIS 421992 administeredsubcutaneously using a 10 μL micro syringe (Hamilton) and a 33-gaugeneedle. In all cases, the injections were administered at P1 and the RNAwas assayed at P8. The RNA splicing patterns in the various tissuesafter administering ISIS 421992 in neonate mice were then observed. Theresults of the ICV administration are presented in FIG. 2B and Table 6.Multiple lanes for each condition represent independent experiments. ICVadministration primarily resulted in increased full-length IKBKAP mRNAin the brain and spinal cord, with moderate effects in the peripheraltissues, whereas subcutaneous administration primarily affectedexpression in the liver, skeletal muscle, and heart with moderateeffects in the CNS (FIGS. 2C, 2F, and Table 7). The plot shows inclusionpercentages of IKBKAP exon 20 in different tissues from five independentICV or subcutaneous injections.

TABLE 6 Percent inclusion of exon 20 in human IKBKAP mRNA levels afterICV administration to neonatal Tg mice Treatment Dose (μg) % PBS — 7ISIS 421992 2.5 13 5 18 10 34 20 36 30 48

TABLE 7 Percent inclusion of exon 20 in human IKBKAP mRNA levels indifferent tissues of neonatal Tg mice s.c. ICV Control injectionadministration Brain 12 18 50 Spinal Cord 9 19 47 Liver 40 72 44 Heart41 58 40 Muscle 29 60 33 Kidney 32 36 33

We claim:
 1. A compound comprising a modified oligonucleotide consistingof 8 to 30 linked nucleosides and having a nucleobase sequencecomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of an IKBKAP transcript.
 2. The compound of claim1, wherein nucleobase sequence comprises at least 8 contiguousnucleobases complementary to intron 19, intron 20, or exon 20 of anIKBKAP transcript.
 3. The compound claim 1, wherein the modifiedoligonucleotide is 12 to 20 nucleosides in length.
 4. The compound claim3, wherein the modified oligonucleotide is 15 nucleosides in length. 5.The compound of claim 4 having a nucleobase sequence comprising at least9 contiguous nucleobases complementary to a target region of equallength of an IKBKAP transcript.
 6. The compound of claim 5, wherein themodified oligonucleotide comprises at least one modified nucleoside. 7.The compound of claim 6, wherein at least one modified nucleosidecomprises a modified sugar moiety.
 8. The compound of claim 7, whereinat least one modified sugar moiety is a 2′-substituted sugar moiety. 9.The compound of claim 8, wherein the 2′-substitutent of at least one2′-substituted sugar moiety is selected from the group consisting of2′-OMe, 2′-F, and 2′-MOE.
 10. The compound of claim 9, wherein the2′-substiuent of at least one 2′-substituted sugar moiety is a 2′-MOE.11. The compound of claim 7, wherein at least one modified sugar moietyis a bicyclic sugar moiety.
 12. The compound of claim 11, wherein atleast one bicyclic sugar moiety is LNA or cEt.
 13. The compound of claim11, wherein at least one bicyclic sugar moiety is cEt.
 14. The compoundof claim 11, wherein at least one bicyclic sugar moiety is LNA.
 15. Thecompound of claim 7, wherein at least one sugar moiety is a sugarsurrogate.
 16. The compound of claim 15, wherein at least one sugarsurrogate is a morpholino.
 17. The compound of claim 15, wherein atleast one sugar surrogate is a modified morpholino.
 18. The compound ofclaim 6, wherein at least one internucleoside linkage is a modifiedinternucleoside linkage.
 19. The compound of claim 18, wherein eachinternucleoside linkage is a modified internucleoside linkage.
 20. Thecompound of claim 19, wherein the modified internucleoside linkage isphosphorothioate.